The discovery of new compounds which exhibit an affinity for biological receptors holds the promise of providing agents which have useful bioactive properties. Biological receptors of interest include, for example, enzymes, antibodies, oligonucleotides, cell surface receptors, etc. A molecule which binds to a biological target can often be expected to display bioactive properties such as pharmacological, herbicidal or pesticidal activity. In the absence of a bioactive response, the affinity of an agent for a biological target is itself a useful property which can be used to probe, for example, the nature of enzymatic reactions, membrane transport of ions, cellular recognition, antibody activity, etc. Until quite recently, the discovery of useful bioactive molecules was either serendipitous or it occurred by the serial synthesis and screening of a large number of potentially active compounds.
The serial synthesis and screening of individual organic molecules which have a potential for pharmacological or other activity is a laborious and time-consuming task. The search for compounds with desirable biological properties begins with the identification of one or more lead compounds which bind to a biological target such as a receptor, a pathogen or an enzyme. To identify a lead compound, numerous compounds are synthesized in an effort to define structural features essential for the desired bioactivity. Once a lead compound has been identified, the structure of the lead compound is varied, in a somewhat orderly fashion, in an attempt to optimize its activity. In general, it is necessary to synthesize and evaluate many compounds in both the lead identification and the optimization steps. Each synthesized compound must be purified, characterized and screened for activity. Recent advances in combinatorial methods of synthesis have considerably simplified the drug discovery process and have improved the means by which large numbers of organic compounds can be synthesized and screened for activity.
Combinatorial synthesis is a high-throughput synthesis technique which has been used to rapidly produce and screen libraries of up to tens of thousands of compounds. A very wide range of chemistry types are amenable to library generation. For example, combinatorial methods have been used to synthesize libraries of oligomeric compounds such as, for example, peptides (Pirrung, et al., U.S. Pat. No. 5,143,854), peptoids (Simon, R. J., et al., Proc. Natl. Acad. Sci. U.S.A., 91:11138-11142 (1994)), and oligodeoxynucleotides (Fodor, et al., PCT Publication No. WO 92/10092). Combinatorial methods have also been used to synthesize non-oligomeric compounds such as, for example, benzodiazepines (Bunin, et al., J. Am. Chem. Soc., 114:10997-10998 (1992)), diketopiperazines (Gordon, D. W., et al., BioMed. Chem. Lett., 5:47-50 (1995)) and pyrrolidines (Murphy, M. M., et al., J. Am. Chem. Soc., 117:7029-7030 (1995)).
Combinatorial libraries have been synthesized both in solution and using solid supports. See, for example, Thomson, L. A., et al., Chem. Rev., 96:555-600 (1996). The principal advantage of a solution-phase approach is that a method does not need to be developed to attach the initial starting material onto the support or to cleave the final product from the support. In spite of these advantages, the majority of the published work on combinatorial libraries has utilized solid-phase synthesis. This is largely due to the relative ease of purifying the support-bound compounds away from excess reagents.
The solid-phase assembly of combinatorial libraries relies on a solid support which bears on its surface a large number of reactive functional groups to which the first component ("building block") of the putative library is attached. The solid support and the attached building block are then contacted with a second building block and any reagent necessary to react the first and second building blocks. The excess second building block and the reagents are removed by washing the solid support with an appropriate solvent. These steps are repeated with additional building blocks and reagents until the library of desired compounds is assembled.
Solid supports made of a variety of materials have been used in assembling combinatorial libraries. A solid support can be manufactured of virtually any material which remains chemically inert and insoluble during the various reaction cycles required to assemble the library. Commonly used solid supports include polystyrene cross-linked with 1-2% divinylbenzene (Merrifield, R. B., J. Am. Chem. Soc., 85:2149-2154 (1963)), aminomethylated polystyrene resin (Plunkett, M. J., et al., J. Am. Chem. Soc., 117:3306-3307 (1995)) and amino-functionalized poly(ethylene glycol)-grafted polystyrene resin (Baldwin, J. J., et al., J. Am. Chem. Soc., 117:5588-5589 (1995)).
Although methods exist in the art for the surface modification of both siliceous (e.g., glass, quartz, mica, etc.) and plastic surfaces with reactive functional groups, these various techniques have yet to be applied to the modification of optical fibers to allow their use as solid supports for combinatorial libraries. The use of optical fibers as solid supports has numerous advantages. The optical fiber can be used to deliver electromagnetic energy in the form of, for example, light, heat or a combination thereof to the reactants on the surface of the fiber. Further, spectroscopic data available via use of the functionalized optical fibers allows a combinatorial library of compounds to be screened for a desirable characteristic while the compounds remain bound to the optical fiber.
The use of optical fiber strands for medical, biochemical and chemical analytical determinations has undergone rapid development. The use of optical fibers for such purposes and techniques is described by Milanovich, et al., "Novel Optical Fiber Techniques for Medical Application", Proceedings of the SPIE 28th Annual International Technical Symposium on Optics and Electro-Optics, Vol. 494, 1980; Seitz, W. R., "Chemical Sensors Based on Immobilized Indicators and Fiber Optics", in C.R.C. Critical Reviews in Analytical Chemistry, Vol. 19, 1988, pp. 135-173; Wolfbeis, O. S., "Fiber Optical Fluorosensors in Analytical Chemistry", in MOLECULAR LUMINESCENCE SPECTROSCOPY, METHODS AND APPLICATIONS, Schulman, S. G., Ed., Wiley and Sons, New York. The optical fiber strands are typically glass or plastic extended rods having a small cross-sectional diameter. When light energy is projected into one end of the fiber strand, the angles at which the various light rays strike the surface are greater than the critical angle; and such rays are "piped" through the strand's length by successive internal reflections and eventually emerge from the opposite end of the strand. When the individual strands are bundled together, the collection is referred to as an "imaging fiber." An imaging fiber can comprise several thousand individual 3-4 .mu.m-diameter optical fibers melted and drawn together in a coherent manner such that an image can be carried from one end to the other. See, for example, Chigusa, Y., et al., Optoelectronics, 1:203-216 (1986); Mogi, M., et al., Proc. SPIE-Int. Soc. Opt. Eng., 1067:172-181 (1989). Typically, bundles of these strands are used collectively in a variety of different applications. One application of particular interest resides in the field of optical sensors.
For making an optical fiber into an optical sensor, one or more light absorbing fluorescent dyes are located at the distal end of the optical fiber. For example, Walt, et al. (U.S. Pat. No. 5,320,814) teaches optical sensors which comprise, at their distal ends, a polymeric substance of predetermined chemical composition and a dye compound which is disposed in admixture with the polymeric substance. The admixture is formed by the photopolymerization of an acryloyl derivative of a fluorescent dye. The admixed dye compound absorbs light energy and, in the presence of an analyte, generates a modified spectral response which is detected at the proximal tip of the fiber. Importantly, Walt, et al. neither teaches nor contemplates the assembly of an orderly combinatorial array of compounds using optical fibers as a solid synthesis support. Further, Walt, et al. neither teaches nor contemplates screening a combinatorial library of individual compounds, immobilized on an optical fiber, by using the fiber to spectroscopically detect interactions between the library compounds and a fluorescent-labeled analyte.
Methods of detecting optical changes at the distal tip of individual fibers of an optical imaging fiber are known in the art. One such means of detection utilizes an imaging fiber in association with an epifluorescence microscope and a charge-coupled device (CCD) camera. Bronk, K. S., et al., Anal. Chem., 67:2750-2757 (1995). The apparatus described by Bronk, et al. allows for the simultaneous imaging and chemical sensing of a sample.
In spite of the recent advances in the use of optical fibers as chemical sensors, a method has yet to be produced for the use of optical fibers for the synthesis and screening of combinatorial libraries of compounds. Chemical synthesis of a compound library on an optical fiber would allow electromagnetic energy such as light and/or heat to be supplied to the reactants on the surface of the optical fiber. Application of these forms of energy to the reactants facilitates those reactions requiring the input of light or heat energy.
Combinatorial libraries can be assembled by functionalizing individual optical fibers arranged in an imaging fiber with a different component of the library. Following the assembly of the library, the library compounds can be screened for desirable properties such as their binding to, for example, a fluorescent labeled biomolecule. The screening for binding can be conducted while the compound is attached to the optical fiber and the binding can be detected using the optical fiber. Also, using the imaging properties of the imaging fiber, the individual fibers to which the fluorescent molecule has bound, can be easily located. Thus, a method utilizing optical fibers for the synthesis and screening of a combinatorial library of compounds would represent a significant addition to the growing arsenal of techniques for the synthesis and screening of these libraries. Quite surprisingly, the instant invention provides such a method.